Abstract 1976: Development of a CD137 receptor occupancy assay to support the phase I/II study of BT7480, a <i>Bicycle</i> tumor-targeted immune cell agonist™ (<i>Bicycle</i> TICA®)
Authors: Heather Cohen, Drasti Kanakia, Johanna Lahdenranta, Punit Upadhyaya, Kristen Hurov, Julia Kristensson, Chintan Jobaliya, Greg Bannish, Adam Cotty, Kevin McDonnell, Sandra Hirschberg, Phil Brandish, Sebastien Hazard, Dominic Smethurst, Nicholas Keen, Stephen J. Blakemore
Journal: Cancer Research (2022)

Abstract Bicycles are fully synthetic constrained peptides with antibody-like affinities that target selectively, readily penetrate tumor tissue, have relatively short half-lives, and can be chemically linked together to generate multifunctional molecules. BT7480 is a Bicycle TICA® being developed as a first-in-class CD137 therapeutic for the treatment of human cancers associated with Nectin-4 expression which is currently being investigated in an ongoing phase I/II clinical trial. Monitoring target engagement for a given therapeutic can be a key factor in recommending the phase II dose. While flow cytometry-based receptor occupancy (RO) assays are commonly used to monitor target engagement in the clinic, a CD137-specific RO assay presents several important challenges that have historically hampered monitoring RO in the clinic including the dynamic expression of CD137 on unstimulated and stimulated T cells, the low frequency of CD137+ cells in human blood and limited reagents to confidently detect CD137+ cells in the presence of CD137-targeting drugs. To address these challenges, a fit-for-purpose 14-plex flow cytometry panel was developed that incorporates a fluorescently labelled CD137-specific binding Bicycle®. This CD137 Bicycle® was shown to directly compete with BT7480 for binding to CD137, but not with fluorescently labelled anti-CD137 antibody, thereby enabling simultaneous detection of various CD137+ immune cell types as well as receptor occupancy by BT7480 in a single blood sample. Panel performance was tested across blood-based sample matrices routinely used in the clinic including EDTA and Cyto-Chex® blood collection tubes and Cell Preparation Tubes (CPT) (n=3 each). CPT were selected as the optimal sample matrix based on sample viability and highest detection of CD137 antibody+ and CD137 Bicycle® + cells. Ex vivo RO assessments in anti-CD3 stimulated and unstimulated healthy human blood, demonstrated dose-dependent detection of CD137 RO by BT7480 and the detection of &amp;gt;1000 CD137+ cells with sample viability &amp;gt;70% (n=5 each). The optimized method and dose-dependent detection of CD137+ cells and RO by BT7480 was further verified in unstimulated lung cancer patient whole blood samples (n=5). Results from this study represent the first report of a clinic-ready CD137 RO assay and the first flow cytometry assay using fluorescently labelled Bicycle® reagents and demonstrate the utility of the Bicycle® CD137 RO assay to monitor target engagement in the BT7480 first-in-human clinical trial. Citation Format: Heather Cohen, Drasti Kanakia, Johanna Lahdenranta, Punit Upadhyaya, Kristen Hurov, Julia Kristensson, Chintan Jobaliya, Greg Bannish, Adam Cotty, Kevin McDonnell, Sandra Hirschberg, Phil Brandish, Sebastien Hazard, Dominic Smethurst, Nicholas Keen, Stephen J. Blakemore. Development of a CD137 receptor occupancy assay to support the phase I/II study of BT7480, a Bicycle tumor-targeted immune cell agonist™ (Bicycle TICA®) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1976.